The present invention relates to the field of plant molecular biology. In particular, it relates to the creation of transformed rice plants via accelerated particles.
Rice, Oryza sativa, is an important food crop that provides more calories than any other cereal on a per hectare basis and is second only to wheat in area harvested worldwide. There is a great deal of interest in new rice varieties, but conventional plant breeding methods have several inherent limitations. Several generations of selfing are required for homozygosity, so breeding programs for new rice varieties may take several years. Genetic traits from wild rice varieties are potentially useful in improving rice resistance to pests, diseases, and adverse environment, but wild rice varieties do not cross well with domestic rice.
Because of the limits of conventional plant breeding, rice has been genetically engineered. Luo and Wu, Plant Molec. Biol. Rep. 6 [3]: 165-174 (1988) and Plant Molec. Biol. Rep. 7 [1]: 69-77 (1989), reported a method of transferring foreign DNA into recently pollinated rice florets which they term xe2x80x9cpollen tube pathwayxe2x80x9d transformation. The pollen tube pathway process involves slicing away the top of the rice floret so that the stigma is cut off and the style has a severed end. A drop of a DNA-containing solution is placed at the cut end of the floret and allowed to flow down the pollen tube. Eventually, seeds are harvested and germinated. Pollen tube pathway transformation, however, has not been repeated by others and does not seem to be reproducible.
Rice protoplasts, the rice plant cell isolated from the cell wall, have been a popular target of genetic engineering. One common transformation method is to incubate the protoplasts with DNA and polyethylene glycol (PEG) and subsequently culture the protoplasts into plants in the presence of a selective agent. For example, Uchimiya et al., Mol Gen Genet: 204-207 (1986), describe PEG-mediated transformation of rice protoplasts to produce the expression of the foreign gene in transformed callus. Other examples of PEG-mediated protoplast transformation are found in Hayashimoto et al., Plant Physiol. 93: 857-863 (1990) and Peng et al., Plant Cell Reports 9: 168-172 (1990).
Rice protoplasts have also been transformed via electroporation. In most electroporation methods, rice protoplasts are mixed with a DNA solution and subjected to an electrical pulse. Similarly to PEG-mediated transformation, the DNA molecules cross the cell membrane and integrate into the cell genome. Recently, Terada and Shimamoto, Mol. Gen. Genet. 2201 389-392 (1990), have described the transformation of rice protoplasts via electroporation to produce mature rice plants that express the foreign gene. Tada et al., Theor. Appl. Genet. 80: 475-480 (1990), and Battraw and Hall, Plant Molec. Biol. 15: 527-538 (1990), have also recently reported transformation of rice via electroporation. Shimamoto, et al., Nature 338: 274-276 (1989), electroporated a hygromycin resistance gene into protoplasts and detected the foreign gene in progeny plants.
Rice has been transformed with a mixture of PEG and electroporation in Yang et al., Plant Cell Reports 7: 421-425 (1988). Transformation was confirmed by enzymatic assays in randomly chosen kanamycin resistant clones.
Transformation methods based on either PEG or electroporation also are limited because they depend on the regeneration of whole transformed rice plants from transformed rice protoplasts. There are many problems and limitations inherent in the protoplast culture regeneration process. Current protoplast regeneration processes result in a high frequency of somaclonal variation, albinism, and sterility. Additionally, and most importantly, not all rice varieties can be regenerated from protoplasts. Rice is grouped into two major groups of varieties, designated Japonica and Indica. Japonica and Indica varieties differ on the basis of their geographical distribution and morphological and physiological elements. Japonica and Indica varieties also differ in their amenability to tissue culture and regeneration methods. In general, Japonica varieties have high callus yield and high regeneration ability, and Indicas have very poor callus growth and poor regeneration potential. Indica varieties are, in general, the most commercially desirable.
The transformed rice plants produced by the procedures discussed above were Japonica varieties. Luo and Wu, Plant Molec. Biol. 7 [1]:69-77 (1989), included an Indica variety in their pollen tube pathway experiments. However, the reported positive transformation results have not been repeated and cannot be confirmed. Datta et al., Biotechnology 8: 736-740 (1990), have established a PEG-mediated transformation protocol for plant regeneration from Indica-type protoplasts. Southern analysis and enzyme assays have proved that the foreign gene was stably integrated and inherited in offspring. This method, however, only works on one specific, noncommercial Indica variety. This is the only reported method performed to date on Indica rice varieties that resulted in transformed plants. In general, while much effort has been expended on developing a reproducible system to regenerate Indica varieties, no such procedure has yet been published.
Rice has been transformed via Agrobacterium infection of mature embryos. The first widely used plant genetic engineering technique was based on the natural ability of the soil-dwelling microorganism Agrobacterium tumefaciens to introduce a portion of its DNA into a plant cell as a part of the normal pathogenic process. If a foreign gene is inserted into the bacteria in certain ways, the Agrobacterium can be used to transfer the foreign gene into a plant. Agrobacterium transformation techniques have been developed for a number of plants, mostly dicotyledonous, but the usefulness of the technique has varied from plant species to species. Agrobacterium-based transformation systems are limited, because they require cell or tissue culture and plant regeneration techniques. Plant lines vary in their amenability to tissue culture and regeneration methods. Monocots, such as rice, are especially poor candidates for Agrobacterium-mediated transformation. However, Raineri et al., Biotechnology 8: 33-38 (1990), report Agrobacterium-mediated transformation of rice tissues, as confirmed by DNA hybridization analysis. The inoculated embryos formed tumorigenic callus tissue. No plants were reported, and it appears that it may be impossible to regenerate plants from these tissues.
One new transformation technique attempts to create a transformed plants by bombarding plant cells or tissues with accelerated particles which carry genetic material. The first indication of the utility of this technique was a demonstration that DNA constructs could be coated onto tungsten particles and accelerated into onion skin, where the genes were transiently expressed. U.S. Pat. No. 4,945,050. A problem in the development of an accelerated particle transformation process is the difficulty of obtaining a germline plant transformation. By the term xe2x80x9cgermline transformationxe2x80x9d it is meant that the germ cells of the plant are transformed in such a way that the progeny of the plant inherit the foreign nucleic acid construct inserted with the particles into the parental plant tissue. Plant genetic transformation has been achieved by the accelerated particle method. U.S. Pat. No. 5,015,580 discloses the germline transformation of soybean plants and plant lines. One method disclosed in that published patent application is accelerating DNA-coated particles into excised embryonic axes of soybean plants. If the bombarded soybean embryonic axes are treated with high cytokinin media, shoots are induced from the treated embryonic axes. When the shoots are cultivated into whole soybean plants, a significant percentage of the plants will have transformed germlines. Similar to particle bombardment, transformation of rice has been attempted with an air gun apparatus. Oard et al., Plant Physiol. 92: 334-339 (1990) report transient gene expression when embryogenic callus of rice is bombarded. For particle-mediated transformation efforts, however, it is far easier to obtain transient activity in callus than it is to obtain germline transformant plants.
The art of plant molecular biology needs an efficient method of creating a transformed Indica rice plant. This method would optimally not depend on protoplast culture and regeneration, should be genotype independent, and should readily create germline transformant plants which produce transgenic progeny. It would also be most desirable if the same procedure was available for Japonica varieties as well.
The present invention is both a method of creating transformed rice plants and transformed Indica rice plants. The method begins with the preparation of copies of a nucleic acid construct. These copies are coated onto biologically inert carrier particles. In one embodiment of the present invention, the nucleic acid-coated carrier particles are physically accelerated toward immature rice embryos. In another embodiment, discs are excised from the meristem of a rice seedling. These discs are bombarded with the nucleic acid-coated carrier particles. Both the bombarded embryos and discs are cultivated to produce shoots. These shoots are cultivated into whole, sexually mature plants. The presence of the nucleic acid construct is verified in either the shoots or the sexually mature plants.
One object of the present invention is to produce a transformed Indica rice plant.
Another object of the present invention is to produce an Indica rice plant with transformed progeny.
An advantage of the method of the present invention is that it is successful in all rice varieties.
Another advantage of the method of the present invention is that it does not depend on protoplast culture or embryogenic suspension culture, the regeneration protocols for which are much more genotype dependent.
Other objects, advantageous, and features of the present invention will become apparent from the following specification when taken in conjunction with the accompanying drawings.